Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, in a large and spacious phagolysosome-like parasitophorous vacuole (PV). The recruitment of membrane required for PV biogenesis is a complex process that is modulated by both host and bacterial factors. Moreover, the lipid content of the PV membrane may confer unique properties that allow pathogen growth. We have shown that the PV membrane is sterol-rich and that pharmacologic inhibition of host sterol metabolism negatively impacts PV generation and pathogen replication. Cholesterol is a major sterol component of mammalian membranes where it provides structural stability, signaling platforms called lipid rafts, and serves as a precursor of secondary messenger molecules. Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24-/- mouse embryonic fibroblasts (MEFs). DHCR24-/- MEFs lack the &#916;24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24-/- MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: C. burnetii, Salmonella enterica serovar Typhimurium, and Chalmydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24-/- MEFs. In contrast, C. burnetii entry, but not attachment, was significantly decreased, suggesting C. burnetii utilizes lipid-raft mediated signaling to gain entry into host cells. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24-/- MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, suggesting cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data indicate cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions. We have identified over 30 Coxiella Dot/Icm Type IVB secretion system (T4BSS) substrates that represent a treasure trove of potential virulence factors. These effector proteins are deliverd directly into the host cytosol where they are predicted to modulate an array of host cell processes that promote pathogen growth. Elucidation of their cellular activities and targets will provide needed information on the Coxiella/host relationship. Coxiella Dot/Icm substrates were initially identified using Legionella as surrogate host. However, by using Coxiella genetic transformation methods developed in our laboratory, we have confirmed Dot/Icm-dependent secretion of effectors by Coxiella. Moreover, with transposon and targeted gene inactivation technologies, we generated icmD, dotA, and dotB mutants that were used to verify that a functional T4BSS is required for productive infection of human macrophages by Coxiella. When ectopically expressed in HeLa cells, effectors traffic to different subcellular sites including recycling endosomes, ubiquitin-rich compartments, the endoplasmic reticulum, plasma membrane, and PV membrane. Genes encoding four proteins that traffic to the PV membrane, termed cvpA (Coxiella vacuolar protein A), cvpB cvpC and cvpD are of particular interest becuase they may modulate membrane fusion events. Indeed, strains containing targeted deletions in cvpA and cvpD show pronounced intracellular growth defects. Ongoing studies are focused on identifying the host cell binding partners of Cvp proteins and the host functions they manipulate. Type IV pili (T4P) are defined virulence factors that function in bacterial adherence, twitching motility, biofilm formation, and secretion. Coxiella encodes homologs of type IV pili (T4P) genes; however, their roles in host interactions are unknown. We examined the function of Coxiella T4P genes by generating strains with deletions in pilB, encoding an ATPase involved in pilus assembly, and pilQ, encoding a secretin that translocates T4P proteins across the outer membrane. Wild type and mutant bacteria show similar adherence and uptake by THP-1 macrophages. However, preliminary data suggest mutants may have an intracellular growth defect. An intracellular biphasic developmental cycle whereby highly resistant small cell variant (SCV) morphological forms are generated from large cell variant (LCV) morphological forms is considered fundamental to Coxiella virulence. Previous work from our lab revealed that the LCV is the replicative form of Coxiella, and that SCV and LCV are compositionally and antigenically different. Further molecular and biochemical analyses of SCV and LCV morphogenesis is necessary to better understand the physiological relevance of Coxiella biphasic development. However, intracellular growth of Coxiella imposes considerable experimental constraints. Therefore, we investigated development in our new host cell-free growth medium, acidified cysteine citrate medium-2 (ACCM-2). Developmental transitions in ACCM-2 are indistinguishable from Coxiella propagated in vivo. The fidelity of Coxiella morphogenesis in ACCM-2 now provides ample pure cell populations for biochemical studies, ultrastructural analyses, and phenotyping.